Prophylactic and therapeutic monoclonal antibodies

ABSTRACT

In this application are described vaccinia monoclonal antibodies. Also provided are mixtures of antibodies of the present invention, as well as methods of using individual antibodies or mixtures thereof for the detection, prevention, and/or therapeutical treatment of vaccinia virus infections in vitro and in vivo.

[0001] This application claims benefit from an earlier filed Provisionalapplication Ser. No. 60/182,066 filed on Feb. 11, 2000.

INTRODUCTION

[0002] Viruses in the family Poxviridae, including vaccinia virus (VACV)and variola virus, are characterized by a large linear double-strandedDNA genome (130-300 kb) packaged in a relatively large virion (˜350×270nm), and a cytoplasmic site of replication (reviewed by Moss, 1996, In“Fields Virology”, D. M. Knipe et al. Eds., vol. 3, pp 2637-2671.Lippincott-Raven, Philadelphia). Assembly of VACV virions begins withcondensation of dense granular material into membrane-wrapped particlescalled intracellular mature virions (IMV). Recent findings indiate theIMV are wrapped by a single membrane (Hollingshead et al., 1999, J.Virol. 73, 1503-1517) rather than a double membrane as previouslyreported. IMV are then enveloped in two additional membranes derivedfrom the trans Golgi to form multiple membrane-warpped particles calledintracellular enveloped virions (IEV) (Schmelz et al., 1994, J. Virol.68, 130-147). IEV are moved, possibly by actin polymerization (Cudmoreet al., 1995, Nature 378, 636-638), to the cell periphery, where theoutermost membrane fuses with the cell plasma membrane, exposing acell-associated eneveloped virion (CEV) (Blasco and Moss, 1991, J.Virol. 65, 5910-5920). CEV are released from the cell as extracellularenveloped virions (EEV), which play a role in long-range spread of thevirus (Payne, 1980, J. Gen. Virol. 50, 89-100). IMV released fromdisrupted cells and EEV are both infectious forms of VACV.

[0003] The primary therapeutic tool for the control and eradication ofinfection with VACV include a live virus vaccine to prevent disease, anda vaccinia immune globulin (VIG) to treat dissminated infections. Theexisting VIG product is derived from human donors who have beenvaccinated with the smallpox vaccine, vaccinia virus. As with all humanproducts, the existing VIG must be tested exhaustively for blood bornehuman pathogens such as human immunodeficiency virus and hepatitus B.Therefore, the existing VIG suffers from several drawbacks including thenecessity for using human volunteers, i.e. the use of a live virus as animmunogen which could cause infectious lesions that scar in healthyindividuals and severe disseminated life-threatening infection inimmunocompromised individuals. And, despite continuous screening of thedonor population to assure consistency which is very expensive, productlots can vary significantly between batches and geographic regions.

[0004] Therefore, there is a need to provide an immune globulincomposition which is safe and precisely defined, and which does not relyon human donors. However, it is not known which components of thevaccinia VIG are important for protection nor how many of the ˜200 genescontained in the vaccinia genome encode proteins that would elicit aprotective response upon passive transfer of monoclonal antibodiesdirected to such proteins.

SUMMARY OF THE INVENTION

[0005] This application satisfies the need mentioned above. Thisapplication describes a vaccinia immunoglobulin composition which canserve as a replacement for the presently used VIG. The vacciniaimmunoglobulin composition of the present invention is composed of oneor more monoclonal antibody against vaccinia antigens defined to beimportant for protection. To identify potential targets for poxvirustherapeutics, we generated and characterized a panel of 400VACV-specific monoclonal antibodies (MAbs) in mice. The monoclonalantibodies were first tested for their ability to neutralize virus andthen were tested for their ability to protect mice from challenge. Twochallenge models were used, one that involves dissemination of the virus(in suckling mice) and a challenge that involves a massive challengedose (by intraperitoneal injection). To our surprise, the ability of theMAbs to inhibit plaque formation by vaccinia virus, a standard assay ofvirus neutralization, did not always predict their protective efficacy.Moreover, the monoclonal antibodies differed in their ability to provideprotection depending on the challenge model.

[0006] We found that the majority of moderately neutralizing monoclonalantibodies were directed against a 34 KDa protein later determined to beD8L which on its own did not provide protection in mice. Anothermonoclonal antibody which was neutralizing did not protect againstchallenge when given alone to mice and was directed against the proteinA27L. Neutralizing MAbs binding to the 29-KDa protein (e.g. MAb-10F5,and MAB-7D11), protected mice against intraperitoneal challenge and werefound to react with the IMV product of the L1R gene first described inWolffe, E. J. et al., 1995, Virology 211, 53-63). Nonneutralizing MAbsbinding to 23 to 28-kDA protein (e.g. MAb-1G10) protected againstchallenge with VACV (strain WR) in suckling mice. The target of MAb-1G10was the EEV product of the A33R gene (Roper et al., 1996, J. Virol. 70,3753-3762).

[0007] The LIR and A33R gene product will be called L1R and A33R,respectively. L1R is an essential myristoylated protein associated withthe IMV membrane and is thought to play a role in IMV attachment orpenetration (Franke et al., 1990, J. Virol. 64, 5988-5996; Ravanello etal., 1993, J. Gen. Virol. 75, 1479-1483; Ichihashi et al., 1994,Virology 202, 834-843; Ravanello and Hruby, 1994, J. Gen. Virol. 75,1479-1483; Wolffe et al., 1995, supra). A33R is a nominally nonessentialglycosylated/palmitated protein that forms dimers and is incorporatedinto the outer membrane of EEV (Payne, 1992, Virology 187, 251-260;Roper et al., 1996, supra). A33R is thought to be involved infacilitating direct cell-to-cell spread via actin-containing microvilli(Roper et al., 1998, J. Virol. 72, 4192-4204). Homologs of L1R and A33Rare present in other Orthopoxviruses, e.g. between VACV and variola, L1Ridentity is 99.6% and A33R is 94.1% (Massung et al., 1994, Virology 201,215-240).

[0008] Therefore, it is an object of the present invention to provide acomposition of one or more monoclonal antibody directed against at leastone, preferably two or more, vaccinia virus antigens. Antigenspreferably include L1R and A33R.

[0009] It is another object of the present invention to providemonoclonal antibodies which protect against vaccinia virus infection andbind to epitopes on L1R and A33R gene products. The monoclonalantibodies described below recognize epitopes on the VACV strainConnaught L1R sequence (Genebank #Af226617) and the Connaught strainA33R gene sequence (Genebank #Af226618). L1R and A33R homologs fromother poxviruses can be used as immunogens to produce monoclonalantibodies which would most likely be protective since the homologs inother poxviruses have high identity with the VACV proteins. Otherpoxviruses include other Orthopoxviruses such as variola virus,monkeypox virus, cowpox virus, Parapoxviruses such as orf virus,paravaccinia virus, and unclassified poxviruses such as Tanapoxvirus,Yabapoxvirus and Molluscum contagiosum.

[0010] It is yet another object of the present invention to provide acomposition comprising humanized monoclonal antibodies of the presentinvention for example anti-L1R antibody, or anti-A33R antibody or amixture thereof, as a vaccinia immunoglobulin replacement. The vacciniaimmunoglobulin replacement may further contain other antibodies specificfor vaccinia antigens shown to be effective for elicitingneutralizing/protective antibodies, for example H3L, D8L, B5R, A27L, andA17L. In addition, MAbs against L1R and A33R homologs from otherpoxviruses can be used alone or in combination with the vaccinia MAbs toprovide a therapeutic and prophylactic composition.

[0011] It is another object of the invention to provide for antibodiesthat are functionally equivalent to the antibodies listed above. Thesefunctionally equivalent antibodies substantially share at least onemajor functional property with an antibody listed above and hereindescribed comprising: binding specificity to L1R and A33R,immunoreactivity in vitro, protection against vaccinia challenge whenadministered prophylactically or therapeutically, competition for samebinding site on L1R and A33R. The antibodies can be of any class such asIgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and othersubclasses known in the art. Further, the antibodies can be produced byany method, such as phage display, or produced in any organism or cellline, including bacteria, insect, mammal or other type of cell or cellline which produces antibodies with desired characteristics, such ashumanized antibodies. The antibodies can also be formed by combining anFab portion and a Fc region from different species.

[0012] It is another object of the present invention to provide formixtures of antibodies according to the present invention, as well as tomethods of using individual antibodies, or mixtures thereof for theprevention and/or therapeutic treatment of vaccinia virus infections invitro and in vivo, and/or for improved detection of vaccinia infections.

[0013] It is yet another object of the present invention to treat orprevent vaccinia virus infection by administering a therapeutically orprophylactically effective amount of one antibody of the presentinvention or a mixture of antibodies of the present invention to asubject in need of such treatment.

[0014] It is another object of the present invention to provide passivevaccines for treating or preventing vaccinia virus infections comprisinga therapeutically or prophylactically effective amount of the antibodiesof the present invention which protect against vaccinia virus, incombination with a pharmaceutically acceptable carrier or excipient.

[0015] It is yet another object of the present invention to provide amethod for diagnosis of vaccinia virus infection by assaying for thepresence of vaccinia in a sample using the antibodies of the presentinvention.

[0016] It is still another object of the present invention to providenovel immunoprobes and test kits for detection of vaccinia virusinfection comprising antibodies according to the present invention. Forimmunoprobes, the antibodies are directly or indirectly attached to asuitable reporter molecule, e.g., and enzyme or a radionuclide. The testkit includes a container holding one or more antibodies according to thepresent invention and instructions for using the antibodies for thepurpose of binding to vaccinia virus to form an immunological complexand detecting the formation of the immunological complex such thatpresence or absence of the immunological complex correlates withpresence or absence of vaccinia virus.

[0017] It is another object of the present invention to provideanti-idiotypic antibodies raised against one of the present monoclonalantibodies for use as a vaccine to elicit an active anti-vacciniaresponse.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

[0019]FIG. 1. Passive transfer of L1R-specific MAb protects againstlethal intraperitoneal challenge with VACV (strain WR). Mice wereinjected with the indicated antibody and then, after 24 hrs, werechallenged with VACV (strain WR). A group of 5 previously immunized mice(tail-scarified) served as positive controls. MAb-7D1is a L1R-specificmouse MAb. MAb-1G10 is a A33R-specific mouse MAb. MAb-8E10 is a negativecontrol mouse MAb.

DETAILED DESCRIPTION

[0020] In the description that follows, a number of terms used inrecombinant DNA, virology and immunology are extensively utilized. Inorder to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

[0021] The term “antibody” is art-recognized terminology and is intendedto include molecules or active fragments of molecules that bind to knownantigens. Examples of active fragments of molecules that bind to knownantigens include Fab and F(ab′)₂ fragments. These active fragments canbe derived from an antibody of the present invention by a number oftechniques. For example, purified monoclonal antibodies can be cleavedwith an enzyme, such as pepsin, and subjected to HPLC gel filtration.The appropriate fraction containing Fab fragments can then be collectedand concentrated by membrane filtration and the like. For furtherdescription of general techniques for the isolation of active fragmentsof antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med.23:1011-1019 (1982). The term “antibody” also includes bispecific andchimeric antibodies.

[0022] The language “monoclonal antibody” is art-recognized terminology.The monoclonal antibodies of the present invention can be prepared usingclassical cloning and cell fusion techniques. The immunogen (antigen) ofinterest, is typically administered (e.g. intraperitoneal injection) towild type or inbred mice (e.g. BALB/c) or transgenic mice which producedesired antibodies, rats, rabbits or other animal species which canproduce native or human antibodies. The immunogen can be administeredalone, or mixed with adjuvant, or expressed from a vector (VEE repliconvector, vaccinia), or as DNA, or as a fusion protein to induce an immuneresponse. Fusion proteins comprise the peptide against which an immuneresponse is desired coupled to carrier proteins, such asβ-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin(KLH), and bovine serum albumin, to name a few. In these cases, thepeptides serve as haptens with the carrier proteins. After the animal isboosted, for example, two or more times, the spleen is removed andsplenocytes are extracted and fused with myeloma cells using thewell-known processes of Kohler and Milstein (Nature 256:495-497 (1975))and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988)). The resulting hybrid cells are then clonedin the conventional manner, e.g. using limiting dilution, and theresulting clones, which produce the desired monoclonal antibodies,cultured.

[0023] Monoclonal antibodies raised against vaccinia antigens L1R, A33R,H3L, D8L, B5R, A27L, and A17L are part of the present invention. Thesemonoclonal antibodies were generated from the vaccinia Connaught vaccinestrain. Other strains of vaccinia are expected to contain sequences atleast 90% identical and which will likely produce antigens capable ofeliciting protective/neutralizing antibodies. Such strains includeIHD-J, Brighton, WR, Lister, Copenhagen, Ankara, Dairen I, L-IPV,LC16M8, LC16MO, LIVP, Tian Tan, WR 65-16, Wyeth. Other homologs havingat least 90% identity exist in other poxviruses in the generaorthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, molluscipoxvirus and Yatapoxvirus which members includevariola major and minor virus, monkeypox virus, camelpox virus,raccoonpox virus, ectromelia virus, sealpox virus, contagious ecthymavirus, canarypox virus, juncopox virus, pigeonpox virus, turkeypoxvirus, penguinpox virus, sheepox virus, goatpox, swinepox virus,buffalopox virus, cowpox virus, rabbit fibroma virus, myxoma virus, andmolluscum contagiosum (genus Molluscipoxvirus) which is 59% identicaland 77% similar to vaccinia (Altschul, S. F. et al. 1997, Nucl. AcidsRes. 25, 3389-3402, fowlpox (genus Avipoxvirus), Yata-tumor like virus(Yatapoxvirus), among others (Fenner, Frank, Poxviruses, In “Virology”B. N. Fields et al., eds. Raves Press, Ltd. New York, 1990, pp.2113-2133). Monoclonal antibodies against homologs from these poxviruseswould likely be protective against challenge with the source ofimmunogen virus.

[0024] The term “epitope” is art-recognized. It is generally understoodby those of skill in the art to refer to the region of an antigen, suchas L1R for example, that interacts with an antibody. An epitope of apeptide or protein antigen can be formed by contiguous or noncontinguousamino acid sequences of the antigen. L1R and A33R, like many proteins,contains many epitopes. The epitopes or peptides recognized by theantibodies of the present invention and conservative substitutions ofthese peptides which are still recognized by the antibody are anembodiment of the present invention. These peptides offer a convenientmethod for eluting the vaccinia antigen bound to the respective antibodyon immunoaffinity columns. For example, when an antibody whichrecognizes the epitope for L1R is used in an immunoaffinity column topurify L1R, the peptide recognized by the antibody can be added to theimmunoaffinity column to elute the L1R. Further truncation of theseepitopes may be possible since antigenic epitopes have been reported tobe represented by as few as five amino acid residues.

[0025] The antibodies described in the Examples below are characterizedin that the antibody binds to the appropriate immunogen as measured byassays such as ELIA, immunoprecipitation, or immunofluorescence. Also,the L1R-specific MAbs must neutralize vaccinia virus as measured byplaque redution neutralization test (PRNT). Any monoclonal antibodyretaining these characteristics is related to the present invention.

[0026] By further mapping of the binding site of the monoclonalantibodies described in this application other peptides useful as avaccine or a therapeutic can be predicted. Therefore, in another aspect,this invention relates to a method for identifying protective antigenicepitopes the method comprising (i) reacting a monoclonal antibodydescribed in this application to different overlapping fragmentsencompassing the complete antigen, (ii) identifying a fragment to whichthe protective antibody binds, (iii) narrowing the region containingsites further by reacting the monoclonal with smaller overlappingfragments encompassing the region identified in (ii), and (iv) choosingpeptides to which the antibody binds as possible antigenic epitopes. Thepeptides can then be assayed for their ability to protect an animal fromdisease, or to reduce the severity of disease.

[0027] The epitopes or peptides on the vaccinia antigen to which themonoclonal antibodies bind can constitute all or part of an eventualactive vaccine candidate. An active vaccine or therapeutic candidatemight comprise these peptide sequences and others. These might bedelivered as synthetic peptides, or as fusion proteins, alone orco-administered with cytokines and/or adjuvants or carriers safe forhuman use, e.g. aluminum hydroxide, to increase immunogenicity. Inaddition, sequences such as ubiquitin can be added to increase antigenprocessing for more effective immune responses.

[0028] The present invention also pertains to hybridomas producingantibodies which bind to an epitope of vaccinia antigens. The term“hybridoma” is art recognized and is understood by those of ordinaryskill in the art to refer to a cell produced by the fusion of anantibody-producing cell and an immortal cell, e.g. a multiple myelomacell. This hybrid cell is capable of producing a continuous supply ofantibody. See the definition of “monoclonal antibody” above and theExamples below for a more detailed description of the method of fusion.

[0029] The present invention still further pertains to a method fordetecting vaccinia in a sample suspected of containing vaccinia. Themethod includes contacting the sample with an antibody which binds anepitope of a vaccinia antigen, allowing the antibody to bind to thevaccinia antigen to form an immunological complex, detecting theformation of the immunological complex and correlating the presence orabsence of the immunological complex with the presence or absence ofvaccinia antigen in the sample. The sample can be biological,environmental or a food sample.

[0030] The language “detecting the formation of the immunologicalcomplex” is intended to include discovery of the presence or absence ofvaccinia antigen in a sample. The presence or absence of vacciniaantigen can be detected using an immunoassay. A number of immunoassaysused to detect and/or quantitate antigens are well known to those ofordinary skill in the art. See Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory, New York 1988 555-612). Suchimmunoassays include antibody capture assays, antigen capture assays,and two-antibody sandwich assays. These assays are commonly used bythose of ordinary skill in the art. In an antibody capture assay, theantigen is attached to solid support, and labeled antibody is allowed tobind. After washing, the assay is quantitated by measuring the amount ofantibody retained on the solid support. A variation of this assay is acompetitive ELISA wherein the antigen is bound to the solid support andtwo solutions containing antibodies which bind the antigen, for example,serum from a vaccinia virus vaccinee and a monoclonal antibody of thepresent invention, are allowed to compete for binding of the antigen.The amount of monoclonal bound is then measured, and a determination ismade as to whether the serum contains anti vaccinia antigen antibodies.This competitive ELISA can be used to indicate immunity to knownprotective epitopes in a vaccinee following vaccination.

[0031] In an antigen capture assay, the antibody is attached to a solidsupport, and labeled antigen is allowed to bind. The unbound proteinsare removed by washing, and the assay is quantitated by measuring theamount of antigen that is bound. In a two-antibody sandwich assay, oneantibody is bound to a solid support, and the antigen is allowed to bindto this first antibody. The assay is quantitated by measuring the amountof a labeled second antibody that can bind to the antigen.

[0032] These immunoassays typically rely on labeled antigens,antibodies, or secondary reagents for detection. These proteins can belabeled with radioactive compounds, enzymes, biotin, or fluorochromes.Of these, radioactive labeling can be used for almost all types ofassays and with most variations. Enzyme-conjugated labels areparticularly useful when radioactivity must be avoided or when quickresults are needed. Biotin-coupled reagents usually are detected withlabeled streptavidin. Streptavidin binds tightly and quickly to biotinand can be labeled with radioisotopes or enzymes. Fluorochromes,although requiring expensive equipment for their use, provide a verysensitive method of detection. Antibodies useful in these assays includemonoclonal antibodies, polyclonal antibodies, and affinity purifiedpolyclonal antibodies. Those of ordinary skill in the art will know ofother suitable labels which may be employed in accordance with thepresent invention. The binding of these labels to antibodies orfragments thereof can be accomplished using standard techniques commonlyknown to those of ordinary skill in the art. Typical techniques aredescribed by Kennedy, J. H., et al.,1976 (Clin. Chim. Acta 70:1-31), andSchurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81:1-40). Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dimaleimide method, and others, all of which areincorporated by reference herein.

[0033] The language “biological sample” is intended to includebiological material, e.g. cells, tissues, or biological fluid. By“environmental sample” is meant a sample such as soil and water. Foodsamples include canned goods, meats, and others.

[0034] Yet another aspect of the present invention is a kit fordetecting vaccinia virus in a biological sample. The kit includes acontainer holding one or more antibodies which binds an epitope of avaccinia antigen and instructions for using the antibody for the purposeof binding to vaccinia antigen to form an immunological complex anddetecting the formation of the immunological complex such that thepresence or absence of the immunological complex correlates withpresence or absence of vaccinia virus in the sample. Examples ofcontainers include multiwell plates which allow simultaneous detectionof vaccinia virus in multiple samples.

[0035] As described in greater detail in the examples, the presentinventors have isolated monoclonal antibodies which bind to at least twodifferent vaccinia virus antigens, L1R and A33R, and display in vitroand/or in vivo vaccinia virus protective properties. Significantly, thereactivity of the MAbs is applicable against a broad variety ofdifferent wild type and laboratory vaccinia strains of different types.

[0036] Given these results, monoclonal antibodies according to thepresent invention are suitable both as therapeutic and prophylacticagents for treating or preventing vaccinia infection in susceptiblevaccinia-infected subjects. Subjects include rodents such as mice orguinea pigs, birds or avian, and mammals, including humans.

[0037] In general, this will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesof the present invention to a susceptible subject or one exhibitingvaccinia infection. Any active form of the antibody can be administered,including Fab and F(ab′)₂ fragments. Antibodies of the present inventioncan be produced in any system, including insect cells, baculovirusexpression systems, chickens, rabbits, goats, cows, or plants such astomato, potato, banana or strawberry. Methods for the production ofantibodies in these systems are known to a person with ordinary skill inthe art. Preferably, the antibodies used are compatible with therecipient species such that the immune response to the MAbs does notresult in clearance of the MAbs before virus can be controlled, and theinduced immune response to the MAbs in the subject does not induce“serum sickness” in the subject. Preferably, the MAbs administeredexhibit some secondary functions such as binding to Fc receptors of thesubject.

[0038] Treatment of individuals having vaccinia infection may comprisethe administration of a therapeutically effective amount of vacciniaantibodies of the present invention. The antibodies can be provided in akit as described below. The antibodies can be used or administered as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to vacciniaantigen, or an antibody capable of protecting against vaccinia virus ina recipient patient, the dosage of administered agent will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition, previous medical history, etc.

[0039] In general, it is desirable to provide the recipient with adosage of antibody which is in the range of from about 1 pg/kg-100pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100mg/kg-500 mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (bodyweight of recipient), although a lower or higher dosage may beadministered.

[0040] In a similar approach, another therapeutic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-L1R oranti-A33R responses (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes,Receptors, and Molecular Mimicry (1988), pp 1-5 and 285-300).

[0041] Likewise, active immunization can be induced by administering oneor more antigenic and/or immunogenic epitopes as a component of asubunit vaccine. Vaccination could be performed orally or parenterallyin amounts sufficient to enable the recipient to generate protectiveantibodies against this biologically functional region, prophylacticallyor therapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support. Aminoacids that are useful for these purposes include: tyrosine, lysine,glutamic acid, aspartic acid, cyteine and derivatives thereof.Alternative protein modification techniques may be used e.g.,NH₂-acetylation or COOH-terminal amidation, to provide additional meansfor coupling or fusing the peptide to another protein or peptidemolecule or to a support.

[0042] The antibodies capable of protecting against vaccinia virus areintended to be provided to recipient subjects in an amount sufficient toeffect a reduction in the vaccinia virus infection symptoms. An amountis said to be sufficient to “effect” the reduction of infection symptomsif the dosage, route of administration, etc. of the agent are sufficientto influence such a response. Responses to antibody administration canbe measured by analysis of subject's vital signs

[0043] A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

[0044] The compounds of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a phamaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16 th ed., Osol, A. ed., MackEaston Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the above-described compoundstogether with a suitable amount of carrier vehicle.

[0045] Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

[0046] Administration of the antibodies disclosed herein may be carriedout by any suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), in ovoinjection of birds, orally, or by topical application of the antibodies(typically carried in a pharmaceutical formulation) to an airwaysurface. Topical application of the antibodies to an airway surface canbe carried out by intranasal administration (e.g., by use of dropper,swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatus for administering respirableparticles of pharmaceutical formulations are well known, and anyconventional technique can be employed. Oral administration may be inthe form of an ingestable liquid or solid formulation.

[0047] The treatment may be given in a single dose schedule, orpreferably a multiple dose schedule in which a primary course oftreatment may be with 1-10 separate doses, followed by other doses givenat subsequent time intervals required to maintain and or reinforce theresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitable treatmentschedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedulessufficient to elicit the desired responses expected to reduce diseasesymptoms, or reduce severity of disease.

[0048] The present invention also provides kits which are useful forcarrying out the present invention. The present kits comprise a firstcontainer means containing the above-described antibodies. The kit alsocomprises other container means containing solutions necessary orconvenient for carrying out the invention. The container means can bemade of glass, plastic or foil and can be a vial, bottle, pouch, tube,bag, etc. The kit may also contain written information, such asprocedures for carrying out the present invention or analyticalinformation, such as the amount of reagent contained in the firstcontainer means. The container means may be in another container means,e.g. a box or a bag, along with the written information.

[0049] The contents of all cited references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

[0050] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

EXAMPLE 1

[0051] Testing L1R- and A33R-specific mouse monoclonal antibodies (MAbs)in vaccinia virus (strain WR) intraperitoneal lethal challenge model.

[0052] A passive transfer experiment was performed to determine if mouseMAbs to either L1R or A33R could protect against a lethalintraperitoneal challenge with VACV (strain WR). Groups of 5 BALB/c mice(14-16 weeks old) were anesthetized, bled, and then injected(subcutaneous, behind the base of the neck, using a 1 cc 25G ⅝tuberculin syringe) with 200 ul of either L1R-specific MAb (MAb-7D11ascites fluid) or A33R-specific MAb (MAb-1G10 ascities fluid) or acombination of MAb-7D11 plus MAb-1G10, or a negative control MAb(MAb-8E10, ascites fluid). As positive controls, 5 mice weretail-scarified with 10⁶ pfu of VACV, strain WR, approximately 3 weeksearlier. Twenty-four hours after the antibody injection, the mice werechallenged with 12.5 LD₅₀ (5×10⁸ pfu in 200 ul)of VACV (strain WR).

[0053] The results are shown in FIG. 1. All of the mice injected withMAb-7D11, either alone or in combination with MAb-1G10, survivedchallenge. Only one of the five mice injected with MAb-1G10 survived.All of the mice vaccinated with the negative control MAb-8E10 died. Fourof the five positive control mice lived.

[0054] Thus, these data indicate that the L1R-specific MAb can conferprotection against a lethal challenge with VACV via the intraperitonealroute. The A33R-specific MAb failed to protect against this challenge.

EXAMPLE 2

[0055] Neonatal ICR mice were injected with the indicated antibody, asascitic fluid or protein purified antibody, and then challenged withvaccinia virus (strain IHD-J) by the subcutaneous route. The resultsindicate that A33R-specific MAb-1G10 protectes against vaccinia virus(strain IHD-J) injected by the subcutaneous route whereas theL1R-specific MAb-1F5 does not. Mouse hyperimmune ascitic fluid (HMAF)also protected.

[0056] Thus, these data indicate that the L1R-specific MAb can conferprotection against a lethal challenge with VACV via the intraperitonealroute. The A33R-specific MAb failed to protect against this challenge.This is contrast to this MAbs capacity to protect against a disseminatedVACV (strain IHD-J) infection in suckling mice (Table 1). TABLE 1Survivors/total challenged VACV challenge Antibody transferred Strain(route) PFU (log 10) diluent HMAF Mab-10F5 MAb-1G10 10F5 + 1G10 IHD-J(s.c.) 3.9 0/13 25 ug Ab 25 ug Ab 25 ug Ab 25 ug Ab 9/12 0/12 10/12 9/1125 ul AF 50 ug AB 50 ug Ab 50 ug Ab IHD-J (s.c.) 3.9 0/10 8/10 0/10 9/11 (ea) combined results 0/23 17/22  0/22 19/23 9/11 % survival 0%77% 0% 83% 82%

What is claimed is:
 1. A composition comprising one or more monoclonalantibody directed against a vaccinia virus antigen.
 2. The compositionof claim 1 wherein said vaccinia virus antigen is L1R.
 3. Thecomposition of claim 1 wherein said vaccinia virus antigen is A33R. 4.The composition of claim 2 wherein said composition further comprisesone or more monoclonal antibody directed against vaccinia A33R.
 5. Thecomposition of claim 4 wherein said composition further comprises one ormore monoclonal antibody directed against an antigen chosen from thegroup consisting essentially of: vaccinia H3L, D8L, B5R, A27L and A17L.6. The composition of claim 4 wherein said composition inhibits vacciniavirus infection in a subject in vivo.
 7. The composition of claim 6wherein said subject is avian or mammalian.
 8. The composition of claim4 wherein said composition ameliorates symptoms of vaccinia virusinfection when said composition is administered to a subject afterinfection with vaccinia virus.
 9. The composition of claim 8 whereinsaid subject is avian or mammalian.
 10. The composition of claim 2wherein said monoclonal antibody immunoprecipitates L1R in vitro. 11.The composition of claim 3 wherein said monoclonal antibodyimmunoprecipitates A33R in vitro.
 12. A therapeutic composition forameliorating symptoms of vaccinia virus infection comprising thecomposition of claim 4, and a pharmaceutically acceptable excipient. 13.A passive vaccine against vaccinia virus infection comprising thecomposition of claim
 4. 14. An anti-vaccinia composition, comprising oneor more monoclonal antibodies, wherein at least two of said monoclonalantibodies are directed against L1R and A33R, in an amount effective forinhibiting vaccinia virus infection, and a pharmaceutically acceptablecarrier.
 15. A method of treating vaccinia virus infection comprisingadministering to a patient in need of said treatment an effective amountof a composition according to claim
 4. 16. The composition according toclaim 1 wherein said vaccinia virus antigen is chosen from the vacciniastrain Connaught, IHD-J, Brighton, WR, Lister, Copenhagen, Ankara,Dairen I, L-IPV, LC16M8, LC16MO, LIVP, Tian Tan, WR 65-16, Wyeth.
 17. Apoxvirus monoclonal antibody composition comprising monoclonalantibodies against a homolog of a vaccinia antigen chosen from the groupconsisting of L1R and A33R, said poxvirus chosen from the groupconsisting of: orthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus,leporipoxvirus, suipoxvirus, molluscimpoxvirus, and yatapoxvirus.